Claudin family is the family of cell membrane proteins of approximately 23 kD in molecular weight which have four transmembrane domains and constitute tight junctions. The Claudin family includes 24 members in humans and mice, and each member of the Claudins is known to exhibit a very unique expression pattern depending on each epithelial cell type (Non-Patent Document 1 (Furuse and Tsukita, TRENDS in Cell Biology 2006, 16: 181); Non-Patent Document 2 (Wilcox, et al., Cell 2001, 104: 165); Non-Patent Document 3 (Rahner, et al., GASTROENTEROLOGY 2001, 120: 411); and Non-Patent Document 4 (Morita, et al., Proc. Natl. Acad. Sci. USA 1999, 96: 511)). In the sheet of epithelial cells, a mechanism works to prevent substances from leaking (diffusing) in the intercellular spaces, and cell-cell adhesion systems called tight junctions have been shown to really play a central role as a “barrier” in the mechanism to prevent leakage.
Non-Patent Document 5 (Hewitt, et al., BMC Cancer 2006, 6: 186) or Patent Document 1 (WO 2003/088808) or the like has unveiled the high expression of human CLDN6 transcripts in cancer. Moreover, Non-Patent Document 6 (Osanai, et al., Cancer Sci. 2007, 98: 1557) and Non-Patent Document 7 (Azadeh Arabzadeh, et al., BMC Cancer 2007, 7: 196) contain a mention to human and mouse CLDN6 expressions at protein levels in cancer. Non-Patent Document 6 has demonstrated CLDN6 expression by western blot analysis using a breast cancer cell line MCF7. This document has claimed, as described in the title, that epigenetic silencing of human CLDN6 in the breast cancer cell line promotes anchorage-independent growth of the cancer cells. Non-Patent Document 6 discloses that in the MCF7 cell line, the expression of human CLDN6 serving as a tumor suppressor gene is decreased due to the partial methylation of the promoter region, resulting in reduced apoptotic sensitivity and the diminished ability to form colony, and this decreased expression also causes increase in cancer cell invasiveness and in metalloproteinase activity and the enhanced ability of the cancer cells to migrate and thus contributes to the malignant alteration of cancer.
However, the western blot conducted in Non-Patent Document 6 on human CLDN6 in MCF7 cells is meant to be an experiment to confirm whether the system of siRNA knockdown of human CLDN6 functions. Thus, this document has made no mention of antibodies as materials used or of methods. Moreover, the experiment is not aimed at examining the degree of change in the expression level of human CLDN6 proteins in the breast cancer cell line MCF7 compared with normal tissues. The authors of Non-Patent Document 6 have cited therein the earlier literature Non-Patent Document 8 (Quan and Lu, Carcinogenesis 2003, 24: 1593) and stated that further study was performed based on the description of Non-Patent Document 8. This Non-Patent Document 8 discusses human CLDN6 serving as a tumor suppressor gene for breast cancer because the mRNA expression of human CLDN6 is decreased in breast cancer cell lines BT-474 and MCF7 compared with normal mammary gland epithelial cells. Specifically, in Non-Patent Document 6, the study has been conducted based on the idea that the expression of human CLDN6 proteins is decreased in the breast cancer cell line MCF7 compared with normal mammary glands, and this document has concluded that epigenetic silencing of human CLDN6 in the breast cancer cell line promotes anchorage-independent growth of the cancer cells.
Moreover, Non-Patent Document 7 is a document aimed only at examining, by immunohistochemical staining, change in the expression patterns of several mouse Claudin proteins including mouse CLDN6 proteins in the tumors of mice developed by DMBA/TPA administration-induced chemical carcinogenesis. This document has stated that mouse CLDN6 is expressed in “suprabasal compartment” even in normal mice.
Regarding anti-CLDN6 antibodies, a monoclonal antibody has not been reported yet which allows human CLDN6 on cell membrane surface, i.e., human CLDN6 present in a native form on cell membrane surface, to be recognized by a method such as flow cytometry.
[Patent Document 1] WO2003/088808
[Non-Patent Document 1] Mikio Furuse and Shoichiro Tsukita: Claudins in occluding junctions of human and flies. TRENDS in Cell Biology 2006, 16: 181
[Non-Patent Document 2] Edward R. Wilcox, Quianna L. Burton, Sadaf Naz, Saima Riazuddin, Tenesha N. Smith, Barbara Ploplis, Inna Belyantseva, Tamar Ben-Yosef, NikkiA. Liburd, Robert J. Morell, Bechara Kachar, Doris K. Wu, Andrew J. Griffith, Sheikh Riazuddin, and Thomas B. Friedman: Mutations in the Gene Encoding Tight Junction Claudin-14 Cause Autosomal Recessive Deafness DFNB29. Cell 2001, 104: 165[Non-Patent Document 3] Christoph Rahner, Laura L. Mitic, and James M. Anderson: Heterogeneity in Expression and Subcellular Localization of Claudin 2, 3, 4, and 5 in the Rat Liver, Pancreas, and Gut. GASTROENTEROLOGY 2001, 120: 411[Non-Patent Document 4] Kazumasa Morita, Mikio Furuse, Kazushi Fujimoto, and Shoichiro Tsukita: Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc. Natl. Acad. Sci. USA 1999, 96: 511[Non-Patent Document 5] Kyle J Hewitt, Rachana Agarwal and Patrice J Morin: The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer 2006, 6: 186[Non-Patent Document 6] Makoto Osanai, Masaki Murata, Hideki Chiba, Takashi Kojima and Norimasa Sawada: Epigenetic silencing of claudin-6 promotes anchorage-independent growth of breast carcinoma cells. Cancer Sci 2007, 98: 1557[Non-Patent Document 7] Azadeh Arabzadeh, Tammy-Claire Troy and Kursad Turksen: Changes in the distribution pattern of Claudin tight junction proteins during the progression of mouse skin tumorigenesis. BMC Cancer 2007, 7: 196[Non-Patent Document 8] Chengshi Quan and Shi-Jiang Lu: Identification of genes preferentially expressed in mammary epithelial cells of Copenhagen rat using subtractive hybridization and microarrays. Carcinogenesis 2003, 24: 1593[Non-Patent Document 9] Kohls M D, Lappi D A: Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 2000, 28 (1): 162[Non-Patent Document 10] Nimmerjahn F, Ravetch J V.: Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science. 2005, 310: 1510[Non-Patent Document 11] Nimmerjahn F, Ravetch J V.: Fcγ Receptors: Old friends and new family members. Immunity. 2006, 24: 19